She has also offered to draw up a joint submission with the UK government in an effort to clarify the issue - although that has been rejected by Scottish Secretary Michael Moore, who said it was up to SNP ministers to set out detailed independence plans.

The Scottish government, which is staging an independence referendum in 2014, argues that, in the event of a 'yes' vote, Scotland would remain part of the EU and negotiate its membership terms "from within".

Opponents pounced on comments made in December by the president of the European Commission, Jose Manuel Barroso, who said: "If one part of a country - I am not referring now to any specific one - wants to become an independent state, of course as an independent state it has to apply to the European membership according to the rules - that is obvious."

It may well now prove much more difficult for the Prime Minister and the Tories to challenge the Scottish Nationalists upon the issue of EU membership.

By Brian TaylorPolitical editor, Scotland

The commission's letter to Ms Sturgeon stated: "The European Commission has not commented on any specific situation in relation to any member state and will continue to refrain from doing so."

It added that, "the European Commission would only be able to express its opinion on the legal consequences under EU law of a specific situation upon request from a member state detailing a precise scenario," and that, in the absence of such a scenario, Mr Barroso would "not be in a position to usefully discuss this matter further with you".

Commenting on the letter, Ms Sturgeon wrote on her blog: "The commission now maintains that the Barroso comments, widely reported before Christmas, were not actually meant to be specifically about Scotland at all.

"That is helpful and effectively reasserts the commission's previous position of neutrality on this issue."

The deputy first minister also said it was a "pity" that her request for a meeting was declined, given there had been a number of "significant interventions" in the debate, from figures including Sir David Edward, formally of the Court of Justice, and Prof David Scheffer, former counsel to the US representative to the United Nations.

'Dangerous dynamic'

On the issue of an EU referendum, Mr Salmond said the Prime Minister was trying to appease Eurosceptics in his own party at the same time as depicting himself as a reformer with the interests of the EU at heart.

The first minister added: "The Westminster parties have consistently claimed that a referendum on Scotland's independence causes uncertainty.

"It is now clear the persistent undercurrent of Tory Euroscepticism poses the biggest threat to Scotland's position in the EU and has now helped to hole below the waterline the baseless scaremongering of Alistair Darling and the rest of the No campaign."

Speaking during question time in the Commons, Mr Cameron said: "The Scottish Nationalists, in my view misguidedly, want to leave the United Kingdom as it is.

Media captionDavid Cameron was quizzed on the independence referendum in the Commons

"I will be arguing, and, I think right across the house, will be arguing that Scotland should stay in the United Kingdom.

"What I want to see in Europe is a changed Europe - then we ask the people."

Welsh First Minister Carwyn Jones accused Mr Cameron of introducing a "dangerous dynamic" to the independence referendum, arguing the promise of a vote on Britain's relationship with the European Union was "a gift" for the SNP.

"One of the arguments that's been used in Scotland is that an independent Scotland would have to apply to be members of the EU," Mr Jones told BBC Wales.

"Well now they will argue that the UK is on the way out of Europe anyway, so next year the argument will be, should Scotland be part of Europe or part of Britain?"